目的 研究白头翁皂苷D对结肠癌上皮间质转化（epithelial-mesenchymal transition，EMT）的影响，并基于无翅型MMTV整合位点家族（wingless type MMTV integration site family，Wnt）/β-连环蛋白（β-catenin）信号通路研究其作用机制。方法 通过MTT细胞增殖实验研究白头翁皂苷D对人结肠癌LoVo细胞的影响，细胞划痕实验研究白头翁皂苷D对LoVo细胞迁移的影响，Transwell细胞侵袭实验研究白头翁皂苷D对LoVo细胞侵袭的影响，锚定非依赖性克隆形成实验研究白头翁皂苷D对LoVo细胞恶性转化的影响；采用实时荧光定量聚合酶链式反应（real-time fluorescent quantitative polymerase chain reaction，qRT-PCR）检测LoVo细胞EMT相关基因和Wnt/β-catenin信号通路基因的mRNA水平，采用Western blotting法检测LoVo细胞EMT相关蛋白和Wnt/β-catenin信号通路蛋白的表达水平。结果 白头翁皂苷D显著抑制LoVo细胞增殖活性、迁移、侵袭和克隆形成，与对照组比较，白头翁皂苷D可显著降低EMT相关的波形蛋白（Vimentin）和蜗牛蛋白（Snail）的mRNA和蛋白表达（P＜0.05、0.01）、升高E-钙黏蛋白（E-cadherin）的mRNA和蛋白的表达（P＜0.01），降低Wnt/β-catenin信号通路中Wnt3a、β-catenin、T细胞因子4（T-cell factor 4，TCF4）和原癌基因（myelocytomatosis viral oncogene homolog，c-Myc）的mRNA和蛋白表达（P＜0.05、0.01），升高糖原合成激酶-3β（glycogen synthase kinase-3β，GSK-3β）的mRNA和蛋白表达（P＜0.05、0.01）。白头翁皂苷D抑制LoVo细胞迁移和调节E-cadherin与Vimentin表达的作用可被Wnt/β-catenin信号通路抑制剂IWR-1协同增强，也被Wnt/β-catenin信号通路激动剂SKL2001拮抗减弱。结论 白头翁皂苷D可抑制结肠癌的EMT，其机制在于抑制Wnt/β-catenin信号通路活性。

Objective To investigate the effects of Pulsatilla saponin D (PSD) on the epithelial-mesenchymal transition (EMT) of colorectal cancer, and explore its action mechanisms based on the wingless type MMTV integration site family (Wnt)/β-catenin signaling pathway. Methods Effect of PSD on the cell viability of LoVo cells was determined with the MTT assay. Effect of PSD on the migration of LoVo cells was assessed with the scratch wound healing assay. Effect of PSD on the invasion of LoVo cells was examined with the Transwell invasion assay. Effect of PSD on the malignant transformation of LoVo cells was appraised with the anchorage-independent colony formation assay. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) analysis was carried out to detect the effect of PSD on the mRNA levels of EMT-related genes and the genes of Wnt/β-catenin signaling pathway in LoVo cells. Western blotting was performed to measure the effect of PSD on the expression levels of EMT-related proteins and the proteins of Wnt/β-catenin signaling pathway in LoVo cells.Results PSD significantly inhibited the proliferation activity, migration, invasion and colony formation of LoVo cells. Compared with the control group, PSD could reduce the mRNA and protein levels of EMT-related Vimentin and Snail (P < 0.05, 0.01), increased the mRNA and protein levels of E-cadherin (P < 0.01). It decreased the mRNA and protein levels of Wnt3a, β-catenin, T-cell factor 4 (TCF4), and myelocytomatosis viral oncogene homolog (c-Myc) in Wnt/β-catenin signaling pathway (P < 0.05, 0.01), increased the mRNA and protein levels of glycogen synthase kinase-3β (GSK-3β) (P < 0.05, 0.01). Furthermore, the inhibition of cell migration and the regulation of the expressions of E-cadherin and vimentin in LoVo cells by PSD were synergized by Wnt/β-catenin signaling pathway inhibitor IWR-1, and antagonized by Wnt/β-catenin signaling pathway activator SKL2001. Conclusion PSD may suppress the EMT of colorectal cancer, potentially by inhibiting the activity of Wnt/β-catenin signaling pathway.

R285.5

国家自然科学基金资助项目（81573813）;四川省自然科学基金面上项目（2023NSFSC0653）